In studying the role of cell surface proteins in differentiation, the surface proteins of three cell types that differentiated from a clonal stem cell of a rat neurotumor were analyzed utilizing a newly perfected high resolution two-dimensional gel electrophoresis system. It was found that each type had different surface proteins. Based on these observations, I propose to extend these studies to test the hypothesis that cellular differentiation is accompanied by expression of specific cell surface proteins. Surface proteins of T-t locus mutant mouse embryos will be analyzed and compared to those of wild type embryos. The recessive t mutants of homozygous lethal type are uniquely suited for this purpose since (1) they interfere with development of embryos at six specific stages, (2) they interfere with the further differentiation of ectoderm, (3) they are expressed as the surface antigens, and (4) a number of mutant strains are available. If T-t locus genes do code for surface proteins as indicated, each mutant should display unique, qualitative differences by two-dimensional electrophoresis. The proposed experiments will help to prove that these differences are responsible for the abnormalities and the early stages of embryogenesis are each accompanied by a specific T-t locus surface protein. These proteins would appear to play a central role in cellular differentiation of ectoderms. I propose to utilize teratoma lines of T-t locus mutants and compare surface proteins of embryoid bodies from the wild type and mutant teratoma lines. The use of embryoid bodies will make it possible to obtain embryo-equivalent structures of homogeneous genotypes in large quantities whereas in vivo embryos produced by crosses are genetically heterogeneous to T-locus mutants. Approximately 1000-fold increase in sensitivity of the protein detection method would be required to analyze in vivo embryos. By this improvement, one can study embryos at stages different from embryoid body stages. Preliminary experiments have achieved about 40-fold increase and further experiments will be proposed to improve sensitivity.